Metallothioneins (MT) are ubiquitous, cysteine-rich metal-binding protein which are present in most animal tissues. MTs have been associated with [1] essential metal metabolism (zn, Cu); [2] sequestration of environmentally accumulated toxic metals (cd, Hg); [3] scavenging of reactive oxygen species (ROS); and [4] cellular metabolism of electrophilic alkylating agents and metallodrugs (e.g., melphalan and chlorambucil cisplatin and aurothiomalate). Crustacean and mammalian MTs both have two- domain structures in which each domain uses 9-11 cysteine residues to create a metal-thiolate cluster which binds 3-6 metal ions (Zn, Cd, Cu). Our proposal is based on the hypothesis that the two domain structure, coupled with strong preferences for certain reactions in each domain, is the chemical basis that allows different functions to occur simultaneously and with minimal interference. To test this, we will determine the structure of lobster MT-1 by 2D and 3D NMR methods; differences in protein folding between lobster and mammalian MTs indicate that lobster MT is an important structure-function model. We will synthesize peptides of each domain (betaN and betac formed at the N- and C-termini) and reconstitute them with metal ions for kinetic and thermodynamic studies of their reactions. The proclivity of each domain for reactions with metal ions (Cu, Zn, Cd), apoZn-enzymes, electrophiles and metal-chelates and ROS will be compared to the holo-protein reactions. We anticipate that the differential (relative) reactivity of the domains (alpha more beta or beta more alpha) reverses for specific reactions. Some reactions (e.g. metal sequestration) are expected to occur under thermodynamic control and others (e.g. ROS scavenging and many electrophile reactions) under kinetic control. Molecular modeling will be used to provide a structural basis for domain reactions. The results of this proposal should move MT research from the moot question of which is the "real" function of MT to asking how these functions co-exist in a single protein. They will provide a better basis for considering the competing effects of normal metabolic states, pathological conditions and drug therapies that converge via MT-dependent reactions.